Raymond J. St. Leger

Differentiation of species and strains of entomopathogenic fungi by random amplification of polymorphic DNA (RAPD).

Polymerase chain reaction (PCR)-based technology, involving random amplification of polymorphic DNA (RAPD), was used to assess the genomic variability between 24 isolates of deuteromycetous fungi (Metarhizium anisopliae, Metarhizium flavoviride, unidentified strains of Metarhizium and Beauveria bassiana) which were found to infect grasshoppers or locusts. M. flavoviride showed little intraspecific variability in PCR-amplified fragments when compared to M. anisopliae. The high level of variability in PCR-amplified fragments contained within M. anisopliae was similar to the total variability between B. bassiana, M. anisopliae and M. flavoviride, and suggests that M. anisopliae may include a number of cryptic species. Four polymorphic RAPD fragments were used to probe the genomic DNA of the various species and strains. On the basis of these probes the fungi can be grouped into M. flavoviride, M. anisopliae, or B. bassiana. According to PCR-amplified fragments, previously-unidentified Metarhizium strains were characterized as M. flavoviride. There was little evidence that these fungi, all isolated from, or virulent towards, grasshoppers or locusts, showed host-selection in PCR-amplified fragments. Nor was geographical origin a criterion for commonalty based on PCR-amplified fragments. PCR-fragment-pattern polymorphisms and the construction of probes from one or more of these fragments may provide a useful and rapid tool for identifying species and strains of entomopathogenic fungi.

Productionin vitro of appressoria by the entomopathogenic fungusMetarhizium anisopliae

Abstract Germination on complex media induced conidia of the entomopathogenMetarhizium anisopliae to produce infection structures (appressoria and penetration hyphae) when the germ tube contacted a hard surface. The morphology of the infection structures and their rate of formation are very similar to those observed for blowfly cuticle. Differentiation frequencies were greater (more than 70% as compared with less than 40%) on hydrophobic surfaces [Teflon, polyvinyl chloride, polystyrene, polypropylene, polyester (GelBond), aluminum foil] than on hydrophilic surfaces (agarose-coated polyester and cellophane). Differentiation frequencies were similar on both positively and negatively charged surfaces. Differentiationin vitro was stimulated by low levels of complex nitrogenous nutrients. Analysis of one- or multicomponent media suggested that amino acids and the lipid component of epicuticle act in combination with the hydrophobic cuticle surface to stimulate differentiation during pathogenesis. Thigmotropic and chemical stimuli for production of appressoria appear to be translated primarily during the second round of nuclear division because inhibitors of DNA and RNA synthesis do not prevent germination but block differentiation if applied before the second nuclear division. Inhibition of protein synthesis blocked both germination and differentiation.

Cloning and regulatory analysis of starvation-stress gene, ssgA, encoding a hydrophobin-like protein from the entomopathogenic fungus, Metarhizium anisopliae.

The nucleotide (nt) sequence of a starvation-stress gene (ssgA) of the entomopathogenic fungus, Metarhizium anisopliae, and its deduced amino acid (aa) sequence were determined. The primary structure of the SSGA (96 aa; deduced M(r) = 9925; pI = 4.1) protein shares extensive similarities with fungal wall proteins of the hydrophobin class, and the eight Cys residues and putative signal sequences are conserved. Secondary structure predictions suggest an additional resemblance to low-M(r) toxins and agglutinins. Northern (RNA) blot analysis and nuclear run-on assays demonstrated transcriptional control of expression of ssgA during nutrient deprivation and during formation of infection structures. Hybridizations of M. anisopliae genomic DNA indicate that there is only one form of ssgA in the genome.

Synthesis of proteins including a cuticle-degrading protease during differentiation of the entomopathogenic fungusMetarhizium anisopliae

Abstract Studies of polypeptide synthesis thesis utilizing [ 35 S]methionine, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and “Western blot” analyses revealed that a cuticle-degrading protease with a chymoelastase specificity is the major protein product when Metarhizium anisopliae produces infection structures on an artificial surface or in situ during penetration of host cuticle. Production of chymoelastase by infection structures is favored by levels of nutrients insufficient to induce catabolite repression. Addition of readily utilized nutrients, e.g., alanine, caused extensive growth on the host cuticle but repressed penetration and synthesis of chymoelastase. Thus, the pathogenic process involving infection-related morphogenesis and enzyme production occurs only when it is necessary for the pathogen to establish a nutritional relationship with the host. Studies with inhibitors (8-azaguanine and actinomycin D) suggest that control of enzyme production is at the level of transcription.

Use of fluorochromes in the study of fungal cytology and differentiation

This review provides a general overview of a range of fluorochromes available with a brief description of their properties, uses and limitations in the study of fungi and other cells. Attention is also drawn to ways of optimizing visualization of images in the fluorescent microscope as well as to new developments in this field

Pathogenicity and growth of Metarhizium anisopliae stably transformed to benomyl resistance

SummaryThe insect pathogenic hyphomycete Metarhizium anisopliae was transformed to benomyl resistance using pBENA3, a plasmid containing the benA3 allele from Aspergillus nidulans. The transformation rate was 9 transformants/50 μg DNA/2×106 viable protoplasts. Southern hybridization analyses indicated that the plasmid integrated by nonhomologous recombination at multiple loci. The sites of integration differed among transformants. There was no evidence for autonomous plasmid replication in the transformants. Transformants grew at benomyl concentrations up to 10 times that which inhibits wild type, and they were mitotically stable on either selective or non-selective medium or insect tissue. The transformants were pathogenic to the hornworm, Manduca sexta, producing both appressoria and the cuticle-degrading enzyme, chymoelastase, in the presence of 50 μg/ml of benomyl. These studies demonstrate the potential of using transgenic strains of entomopathogenic fungi along with other components of pest control such as fungicides.

The rodlet layer from aerial and submerged conidia of the entomopathogenic fungus Beauveria bassiana contains hydrophobin

The entomopathogenic fungus Beauveria bassiana produces aerial conidia, submerged conidia or blastospores depending on the culture conditions. Rodlets are observed on aerial and submerged conidia but not on blastospores. Cell wall rodlets were removed by sonication and the proteins dissolved with formic acid. A single major 9·7 kDa protein was found in rodlets of aerial and submerged conidia and showed N terminal sequence similarities to the hydrophobin class of fungal proteins. Hydrophobin could not be recovered from blastospores. Oxidation of the hydrophobin with performic acid produced a protein of higher molecular weight (14·0 kDa). The rodlet layer could not be removed from intact conidia by boiling in sodium dodecyl sulphate (SDS) but could be removed with formic acid. Formic acid treated conidia retained the ability to bind to insect cuticles and retained hydrophobicity as indicated by a phase exclusion assay.

Isolation of a cDNA encoding a novel subtilisin-like protease (Pr1B) from the entomopathogenic fungus, Metarhizium anisopliae using differential display-RT-PCR

Reverse transcription differential display PCR (RT-DD-PCR) was used to identify genes that are specifically expressed by Metarhizium anisopliae when it contacts the host insect cuticle. Using a homology-based subtilisin-like protease primer we identified a hitherto unsuspected differentially expressed subtilisin-like protease (Pr1B) encoding gene. The deduced amino acid sequence shows 54% similarity to the well characterized Pr1A subtilisin of M. anisopliae and karyotype analysis revealed that Pr1A and Pr1B are located on separate chromosomes. Like Pr1A, Pr1B is synthesized as a large precursor (1158 nucleotides; deduced molecular mass = 40031 Da) containing a signal peptide, a propeptide and the mature protease (283 aa; deduced molecular mass = 28714 Da). However, Pr1B possesses several substitutions in the highly conserved sequences comprising the active sites of subtilisins. In particular, the substitution of Thr220 by serine is unique to Pr1B. Substitution of Asn155 by glycine is also very unusual, and we discuss the likely effects these changes will have on the catalytic efficiency of Pr1B.

Protein synthesis in Metarhizium anisopliae growing on host cuticle

In vitro protein synthesis using poly(A + )RNA and a two-step gel system for proteins were used in this study on the entomopathogenic fungus Metarhizium anisopliae to provide an estimate of the magnitude of differential protein synthesis and secretion that may be involved in adapting to growth on insect cuticle. Shortly after being transferred to a media containing cockroach cuticle, mRNAs for certain proteins are repressed while a broad array of mRNAs for other proteins is induced. Concurrent with this, a least 42 proteins were secreted into the media in a process which was sensitive to actinomycin D. The majority of these proteins were acidic (pI range 4·2–5·6) and co-migrated with Con-A/peroxidase stained bands indicating that they might be glycoconjugates. Microsequencing of those polypeptides accumulated in large amounts revealed two NH 2 -terminal amino acid sequences from acidic proteins that were highly homologous to those of animal trypsins. The trypsin nature of the two proteins was confirmed using a combination of gelatin-SDS-polyacrylamide gel electrophoresis and enzyme overlay membranes. The NH 2 -terminal sequence of the major basic protein identified it as a know subtilisin-like proteinase (Pr1). A second basic sequence was identified as a carboxypeptidase. No other homologies were found.

Novel GTP-binding proteins in plasma membranes of the fungus Metarhizium anisopliae

We report the existence of several families of GTP-binding proteins in plasma membranes of Metarhizium anisopliae. Two proteins (18.4 and 24 kDa) resemble mammalian Gn-proteins in their being toxin insensitive, binding [alpha-32P]GTP on nitrocellulose blots of sodium dodecyl sulfate (SDS)-polyacrylamide gels, and also in their immunological properties. Four other proteins (31-38.2 kDa) were similar except that they did not bind [alpha-32P]GTP after treatment with sodium dodecyl sulfate. An 18.2 kDa cholera toxin substrate and three toxin insensitive bands (18.6, 18.8, and 24 kDa) are novel proteins antigenically related both to mammalian G-proteins and ras gene products. An additional 23 kDa pertussis toxin substrate (the major G-protein in a crude mycelial extract) reacted strongly with antisera to G-proteins but not with anti-ras serum. Other substrates ADP ribosylated by cholera toxin or botulinum D toxin were immunologically unreactive. Analysis of the structural and functional characteristics of these multiple GTP-binding proteins will promote a better understanding of signal transduction in fungi.